Strange bands and large smear in western blot - (Jul/20/2011 )

I have run SDS-PAGE gels for some times however i am know facing a big problem
since every time i do a new gel and western blot whith the same samples i am
getting completely different results in the wells, because some bands misteriously
disapear or others stain really black as in the following gel.
I don't know what could be wrong i am doing every thing i used to.
I run the gel at 160V for 1:30min and use running buffer at pH 8.3 with 10% SDS,
but when i finish the western blot i dont get even results and i am getting desperate.
I transfer to a PDVF membrane with CAPS with pH 11.
Could someone please give me some advice, i would really apreciate it.

-nanobio-

nanobio on Wed Jul 20 19:21:45 2011 said:

Hello everyone,

I have run SDS-PAGE gels for some times however i am know facing a big problem
since every time i do a new gel and western blot whith the same samples i am
getting completely different results in the wells, because some bands misteriously
disapear or others stain really black as in the following gel.
I don't know what could be wrong i am doing every thing i used to.
I run the gel at 160V for 1:30min and use running buffer at pH 8.3 with 10% SDS,
but when i finish the western blot i dont get even results and i am getting desperate.
I transfer to a PDVF membrane with CAPS with pH 11.
Could someone please give me some advice, i would really apreciate it.

These three are identical samples? What have you done (or changed) since you started getting these weird results?

-casandra-

Thanks for your reply casandra. Yes, they are similar samples, whole cell extracts. I
always do the same conditions but about 2 weeks ago i get a lot of variability in the
western blot, for instance if i do 2 or 3 blots with the same extracts sometimes i get
the last well with good bands and no bands in the first or second.

casandra on Wed Jul 20 19:44:50 2011 said:

nanobio on Wed Jul 20 19:21:45 2011 said:

Hello everyone,

I have run SDS-PAGE gels for some times however i am know facing a big problem
since every time i do a new gel and western blot whith the same samples i am
getting completely different results in the wells, because some bands misteriously
disapear or others stain really black as in the following gel.
I don't know what could be wrong i am doing every thing i used to.
I run the gel at 160V for 1:30min and use running buffer at pH 8.3 with 10% SDS,
but when i finish the western blot i dont get even results and i am getting desperate.
I transfer to a PDVF membrane with CAPS with pH 11.
Could someone please give me some advice, i would really apreciate it.

These three are identical samples? What have you done (or changed) since you started getting these weird results?

-nanobio-

nanobio on Wed Jul 20 20:17:17 2011 said:

Thanks for your reply casandra. Yes, they are similar samples, whole cell extracts. I
always do the same conditions but about 2 weeks ago i get a lot of variability in the
western blot, for instance if i do 2 or 3 blots with the same extracts sometimes i get
the last well with good bands and no bands in the first or second.

have you tried preparing fresh reagents esp the antibodies? Anyone else in your lab having the same inconsistent results? You've to do a process of elimination here...

-casandra-

Yes, i prepared fresh reagents and antibodies, Unfortunately nobody else works with these Abs in lab.
Gonna try and do that casandra, eliminate step by step and try to find the problem.

Thans for your help.

casandra on Wed Jul 20 20:37:32 2011 said:

nanobio on Wed Jul 20 20:17:17 2011 said:

Thanks for your reply casandra. Yes, they are similar samples, whole cell extracts. I
always do the same conditions but about 2 weeks ago i get a lot of variability in the
western blot, for instance if i do 2 or 3 blots with the same extracts sometimes i get
the last well with good bands and no bands in the first or second.

have you tried preparing fresh reagents esp the antibodies? Anyone else in your lab having the same inconsistent results? You've to do a process of elimination here...